Hookworms are intestinal nematodes that infect over 1 billion persons worldwide, with a higher prevalence in children than in adults (briefly reviewed in Cecil's Textbook of Medicine, 19th ed., W. B. Saunders Co., 1992, page 2010). These individuals suffer from intestinal hemorrhage as a direct consequence of blood loss caused by the adult hookworms attached to the mucosa. Hookworm disease is most common in tropical and less developed countries, where environmental and socioeconomic conditions including warm, moist soil, lack of public sewage disposal systems and the habit of walking barefoot especially favor transmission. Although other routes of infection are known, such as lactogenic transfer of larvae to infants and use of soiled bedding and clothing (Hotez, P. J., Pediatr. Infect. Dis. J., 8: 516-520 (1989)), infection often occurs when exposed skin maintains contact for several minutes with soil contaminated with parasite eggs containing viable larvae. These penetrate the skin and journey to the lungs to develop into adults that eventually make their way to the upper small intestine, where they attach to the mucosa.
Hookworm disease is due primarly to gastrointestinal blood loss and attendant iron deficiency anemia. Adult worms attached to the mucosa digest ingested blood as well as cause focal bleeding. Each hookworm can suck as much as 0.2 ml of blood per day (Spellman, G. G., and Nossel, H. L., Amer. J. Phys. 220: 922-927 (1971)). This dramatic blood loss can reduce peripheral hemoglobin concentrations to as low as 3 g/100 ml. More commonly, however, blood loss is insidious, and results in chronic iron-deficiency anemia. Thus, in its human host, the adult hookworm functions as a conduit that empties blood into the intestinal tract, producing blood loss on a global scale equivalent to the exsanguination of 1.5 million people per day (Hotez, cited above). Nutritional deficiencies secondary to coexisting conditions that result in low iron stores contribute to morbidity.
The remarkable ability of a single small parasite to cause so much blood loss raises the question of an effective anticoagulating mechanism. Loss of blood from the gastrointestinal tract would be facilitated if the ability of blood to clot were impaired in persons infected with this parasite. Early in this century, researchers observed that extracts of the dog hookworm contained a substance that delayed coagulation of human blood (Loeb, L., and Fleisher, M. S., J. Infect. Dis. 7: 625-631 (1910)). Some fifty years later, it was subsequently noted that hookworm protein, when added to mammalian plasma, markedly prolongs both the prothrombin and partial thromboplastin times (Spellman and Nossel, cited above, and Carroll, S. M., et al., Thromb. Haemostas. 51: 222-227 (1984)).
Although some of this effect has been attributed to a fibrinogenolytic and fibrinolytic protease that degrades fibrinogen (Hotez, P. J., et al., J. Biol. Chem. 260: 7343-7348 (1985)), the exact location in the clotting cascade at which the predominant anticoagulant effect is exerted has not been determined. One investigator reported that extracts of hookworm cephalic glands, while significantly prolonging the prothrombin time, had no appreciable effect on the Stypven-activated factor X clotting time; the anticoagulant was characterized as a protein with a molecular weight between 20,000 and 50,000 daltons (Eiff, J. A., J. Parasitol. 52: 833-843 (1966)). Other investigators, on the other hand, demonstrated that extracts of the whole worms did, in fact, prolong the Stypven time, arguing in favor of the presence of an inhibitor of factor Xa (Spellman and Nossel, cited above).
Blood coagulation, initiated by substances in injured tissues, is propagated by an interlocking network of enzymatic activation, propagation, and control events, the so-called coagulation cascade. These complex reactions ensure that blood coagulation happens quickly and yet remains localized. Blood coagulation results in the formation of a protein scaffolding, the fibrin clot, that controls bleeding and serves as a nidus for subsequent cellular ingrowth and tissue repair. After several days, the fibrin clot is lysed and replaced with a more permanent scaffolding of connective tissue matrix molecules. Abnormalities that result in delay of clot formation or premature lysis of clots are associated with a bleeding tendency.
Coagulation and fibrinolysis involve many blood plasma proteins (see, for example, Table 155-1 in Cecil, cited above, page 1000), with the list growing longer as blood coagulation mechanisms are studied in greater detail. Structural and functional similarities can be employed to group the proteins. For example, one group are zymogens of serine proteases, and hence members of the serine protease family of proteins which includes trypsin, chymotrypsin, elastase, plasmin and cathepsin G. In the coagulation cascade, Factors II, VII, IX, X, XI, XII and protein C are in the serine protease family. These are modified by a vitamin K-dependent posttranslational carboxylation of glutamic acid residues, which allows the proteins to bind calcium and phospholipids and thereby participate efficiently in blood coagulation. Tissue plasminogen activator in the coagulation cascade is also a serine protease. Other proteins are Serine protease inhibitors and hence members of the "serpin" family of proteins, which includes antithrombin III, heparin cofactor II, and plasminogen activator.
Blood coagulation can be initiated by exposure of blood to tissue factor, the so-called "extrinsic system", or by activation of contact factors of plasma, the so-called "intrinsic system". Both of these initiation pathways lead to a common pathway, which results in the elaboration of thrombin, the master coagulation enzyme. Two major coagulation tests mentioned above differentiate these pathways. In the prothrombin time (herein denoted PT) test, tissue factor is added to plasma so that activation proceeds by the extrinsic pathway. In the partial thromoplastin time (herein denoted PTT) test, blood plasma is activated by the intrinsic pathway. The pathways are related somewhat because deficiencies of Factor IX, an intrinsic factor, as well as the factors that follow Factor IX in the intrinsic and common pathways and Factor VII, an extrinsic factor that activates IX and X, are all associated with a bleeding tendency. In contrast, deficiency of Factor XII and prekallikrein, which activates XII, does not cause a bleeding problem.